Table S8: Sample preparation, instrumental analysis, validation parameters and application of sent standard mixture for laboratory coded 8

Laboratory ID / Sample preparation
(sample pre-treatment: pH adjustment, filter pore size, material and trade name, extraction method and conditions, additional clean-up, derivatisation, internal standard…) / Instrumental analysis
(operational parameters: separation and detection condition,…) / Validationparameters
(sensitivity, accuracy, recovery, reproducibility, repeatability,...) / Application of sent standard mixture
500 mL were filtered through 0.7 µm GF/F filter paper from Whatman (UK) and spiked with d4-CP at 5 ng L-1.
Strata X 500 mg-6mL SPE columns (Phenomenex) were loaded onto the Autotrace SPE platform and sequentially conditioned with 12 mLEtOAc, 12 mLMeOH, and 12 mL H2O at a flow rate of 5 mL min-1. The sample lines were pre-cleaned with 25 mL of i-PrOH and 25 mL of H2O. The pre-filtered samples were then loaded at either 4 mL min-1. Once loaded, the sorbent beds of the SPE cartridges were first washed with 12 mL of HPLC grade water followed by a wash with 40% MeOH in water. The cartridges were then transferred to the SPE manifold and dried for 30 minutes. Drying was achieved by securing 100 mL cartridge bodies loaded with anhydrous calcium chloride to the inlet of the Strata X column using SPE column adaptors and drawing air through the stacked cartridges using a vacuum pump.
Each cartridge was then eluted with 2 x 5 mL (1 mL min-1) of EtOAc. The extract was then blown down to dryness with nitrogen using a TurboVap® LV (Biotage, Uppsala, Sweden) set to 40 ⁰C and 10 psi. The residue was dissolved in 3 mL of EtOAc and quantitatively transferred into the reservoir of a conditioned (3x3 mLEtOAc) Florisil® cartridge using a Pasteur pipette; then loaded at 1 mL min-1, with the eluant discarded to waste. The initial test tube was rinsed twice with 2 mL of EtOAc and the rinsing in-turn transferred to the cartridge reservoir and loaded. The Florisil® cartridge was then eluted with 3 x 3 mL of 10% MeOH in EtOAc and the eluant was reduced to dryness, as before, and dissolved in 500 µL mobile phase (5:95 – MeOH/Water both with 0.1% formic acid). / The Accela LC column oven was set to 50 ⁰C. Solvent A was 0.1% formic acid-H2O and solvent B was 0.1% formic acid – MeOH. The mobile phase flow rate was set to 300 µL min-1. The injection volume was 10 µL and the analytes eluted from the column using the following gradient program: mobile phase B - 0 min, 5%; 6 min, 38%; 6.5 min, 100%; 7 min, 100%; 7.5 min, 5%; 12.5 min, 5%; using these conditions IF eluted at approximately 5.34 minutes and CP at 5.74 minutes. To minimise the possibility of MS/MS source contamination from early eluting matrix material, the LC flow was only diverted to the MS/MS between three and eight minutes of the acquisition period. Chromatographic separation was achieved using a HypersilGOLD C18 column (50 x 2.1mm, 1.9 µm diameter particle size) also supplied by ThermoFisherScientific.
The ThermoScientific Quantum Ultra MS/MS was operated in positive electrospray mode using the following settings:
Parameter / HESI
Polarity / Positive
Spray voltage (V) / 3,000
Discharge current (µA) / na
Vaporiser temperature (⁰C) / 350
Sheath gas pressure (arbitrary units) / 30
Ion sweep gas pressure (arbitrary units) / 0
Auxiliary gas pressure (arbitrary units) / 30
Ion transfer capillary temperature (⁰C) / 300
Collision gas pressure (mTorr) / 1
Skimmer offset voltage (V) / -5
MS/MS Ion source parameters
Parameter / HESI
Polarity / Positive
Spray voltage (V) / 3,000
Discharge current (µA) / na
Vaporiser temperature (⁰C) / 350
Sheath gas pressure (arbitrary units) / 30
Ion sweep gas pressure (arbitrary units) / 0
Auxiliary gas pressure (arbitrary units) / 30
Ion transfer capillary temperature (⁰C) / 300
Collision gas pressure (mTorr) / 1
Skimmer offset voltage (V) / -5
SRM transitions and parameters for the investigated analytes
Analyte / Precursor
Ion / Transition
Ion
CP / 261.019 / 139.974
261.019 / 141.968
d4-CP / 265.045 / 139.962
265.045 / 141.984
IF / 261.020 / 92.040
261.020 / 153.953
/ R2 for CP and IF calibration graph produced by Xcalibur™ for solvent standards were typically 0.9995 and 0.992 respectively.
In both cases LODs and LOQs were estimated by averaging the measured S/N (RMS) ratio of the analyte peak in the samples (n=9).
LOD (CP) = 0.04 ng L-1
LOQ (CP) = 0.14 ng L-1
LOD (IF) =0.09 ng L-1
LOQ (IF) =0.29 ng L-1
Recoveries were 59% and 101% for CP and IF, respectively.
Precision was calculated at 1.7 ng L-1 CP and 1.1 ng L-1 IF concentrations and was 7% and 15%, respectively.
Accurracy was determined as intraday and interday accuracy:
CP-intraday: 106%
CP-interday: 109%
IF-intraday: 113%
IF-interday:113% / C: Standard mixture was not used